This proposal focuses on unresolved issues of early B lineage differentiation in mice and, especially, in humans. (Aim 1) A novel model of human B cell development has been identified, the EU12 cell line, in which CD34+/mu heavy chain pro-B cells spontaneously undergo step-wise differentiation to become IgM+/IgD+ B cells. This clone will be used to (i) test the hypothesis that intraclonal V(D)J recombinatorial diversification contributes to the efficient generation of a primary B cell repertoire, (ii) define the changes in gene expression profile during B lineage differentiation, (iii) test the effects of modifying newly-identified and previously-recognized B lineage genes in pro-B cells by sense, antisense and dominant-negative transgenes, and (iv) examine the effects of ligating cell surface receptors (preBCR, BCR, IL7R, CD19, CD32, and CD40) on growth and differentiation of these B lineage cells. (Aim 2) A recently- developed, highly-amplified immunofluorescence method will be used to identify pro-B, pre-B and B cell receptor components on primary B lineage cells to define similarities and differences in the human and mouse B cell differentiation programs. After redefinition of when and where the mu heavy chains, surrogate light chains, kappa/lambda light chains, and Iga/beta are expressed during B lineage differentiation in both species, pro- B, pre-B, and B cell subpopulations will be isolated for comparative analysis of their gene expression profiles and differentiation potential. The latter experiments will incorporate new information obtained in the analysis of differentiation-related changes in the gene expression profile of EU12 cells. (Aim 3) Complementary ex vivo models will be used to elaborate early B lineage differentiation events in mice and humans. An IL-7 producing stomal cell model will be employed to delineate early events in mouse B lineage differentiation. Mouse stromal cells, with and without modification by human stromal cell-derived factor 1 (SDF-1), IL-7 and Fcgamma receptor transgenes, will be employed to evaluate the capacity of human lymphoid progenitors to undergo B lineage differentiation. This comparative analysis will define novel features of B cell differentiation that are likely to have significant clinical implications.